Article pubs.acs.org/Langmuir
AFM Images of the Dark Biocidal Action of Cationic Conjugated Polyelectrolytes and Oligomers on Escherichia coli Lance E. Edens,† Ying Wang,†,‡ David G. Whitten,‡ and David J. Keller*,† †
Department of Chemistry and Chemical Biology and ‡Department of Chemical and Biological Engineering and the Center for Biomedical Engineering, University of New Mexico, Albuquerque, New Mexico 87131, United States ABSTRACT: Polymers and oligomers with conjugated phenylene ethynylene or thiophene ethynylene backbones have been shown to be potent antimicrobials. The mechanisms by which they act have been unclear, though AFM imaging of Escherichia coli cells before and after exposure to two such biocides, PPE-Th polymer and EO-OPE-1(C3), shows their effects on cell surface structure. Dried, unexposed E. coli cells could be imaged at resolution high enough to discern the physical structure of the cell surfaces, including individual porin proteins and their distribution on the cell. Exposure to 30 μg/mL PPE-Th polymer caused major cell surface disruption due to either emulsification of the outer membrane or the formation of polymer aggregates or both. In contrast, exposure to 30 μg/mL EO-OPE-1(C3) oligomer did not cause large-scale membrane disruption but did cause apparent reorganization of the surface proteins into linear arrays or proteinlipid-OPE complexes that dominate on a small scale. E. coli cells were also successfully imaged underwater, allowing a real-time AFM image series as cells were exposed to 30 μg/mL EO-OPE-1(C3). Solution exposure caused the cell surfaces to noticeably increase their roughness over time. These results agree with proposed mechanisms for cell killing by PPE-Th and EO-OPE1(C3) put forth by Wang et al.1 in which PPE-Th kills by large-scale disruption of the outer membrane and EO-OPE-1(C3) kills by membrane reorganization with possible pore formation.
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INTRODUCTION Conjugated polyelectrolytes (CPEs) have been well studied for both their charge-transfer and antimicrobial properties. Recently, Wang et al.1 reported on the ongoing investigation of a series of poly(p-phenylene ethynylene) (PPE)-based polymers and controlled-length oligo-(p-phenylene ethynylene)s (OPEs) with functional side groups that exhibit both dark- and light-activated killing/inactivation mechanisms against a wide range of bacterial species.2−7 In this study, we focus on morphological changes of Escherichia coli cells that result from the dark killing/inactivation activity of two such materials, PPE-Th polymer and EO-OPE-1(C3) oligomer. PPE-Th is a large thienylene-linked PPE polymer with quaternary amine side chains (Figure 1a). It tends to form aggregates in aqueous environments via intra- or interchain stacking of the hydrophobic backbone. EO-OPE-1(C3) is a small oligomer with a linear backbone, charged end groups, and no side chains (Figure 1b). The lack of side chains gives this oligomer needlelike geometry. When these materials absorb light in the visible/near-UV region, low-lying triplet states are formed which can generate singlet oxygen.8 In the dark, the amphiphilic materials interact directly with various cellular targets causing cellular damage and microbial killing.8−10 PPE-Th and EO-OPE-1(C3) are representative of the wider class of polymeric p-phenylene ethynylene (PE) biocides (CPEs) and oligomeric PE biocides (OPEs), respectively. Wang et al.1 has shown that these two biocides have different mechanisms for dark killing of Gram-negative bacteria. EO© 2014 American Chemical Society
Figure 1. Structures of the conjugated polyelectrolyte biocides used in this study.
OPE-1(C3) causes fusion and membrane failure in large unilamellar vesicles (LUVs), along with significant cell envelope disruptions and time-dependent cell cytoplasm release in TEM images of exposed E. coli. In contrast, the PPE-Th polymer causes LUV degradation and, in TEM images, forms large surface debris on bacterial cell envelopes. Longer CPE polymers cause sudden ruptures of LUVs and roughen the outer cell surface with the formation of aggregate clumps. PPEReceived: June 20, 2014 Revised: August 15, 2014 Published: August 18, 2014 10691
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M). The Cell-Tak solution (50 μL) was pipetted onto a cleaned glass coverslip. The sample was allowed to absorb for 1 h (in a closed container with 100% humidity) before being gently rinsed with 0.5 mL of nanopure water. The slides were then thoroughly dried under N2 gas. To deposit cells, a solution containing 108 cells/mL E. coli in distilled water solution was pipetted onto the Cell-Tak-covered slides. The sample was allowed to dry in air until approximately two-thirds of the initial droplet evaporated. The sample was then mounted for imaging in an AFM fluid cell. Before imaging, the fluid cell was flushed with nanopure water to remove loose cells. All fluid images were taken with triangular silicon nitride cantilevers (Veeco model SNL-10 with force constant 0.32 N/m) in a fluid cell containing either nanopure water or 30 μg/mL EO-OPE-1(C3) in water. Standard Veeco imaging software (Nanoscope V531r117) was used to capture and analyze the images. Image Processing. All postcapture image processing was performed using the Veeco Nanoscope V531r1 imaging software.17 All images except those in Figure 5 first underwent an initial plane fit (order 3, no threshold). Then every image was passed through a lowpass filter to smooth feedback noise and artifacts. Images in Figures 2−4 without a scale bar were passed through an additional Spectrum2D (Fourier) filter to enhance the image contrast. To highlight sharper features and eliminate gross surface curvature, a Gaussian highpass was applied to Figure 5d−m. In this article the term real-time zoom means a higher-resolution image created by reducing the AFM scan size while capturing data, but the term software zoom means a higher-magnification image created by interpolation from a previously captured image. A real-time zoom thus has higher resolution while a software zoom has only higher magnification. The histogram in Figure 2d was calculated from the AFM image by highlighting select features by hand (with an approximate threshold height of 8 nm relative to the local background). A binary image was
Th is thought to cause major disruption of the cellular membrane, possibly through an ion-exchange process.1 Atomic force microscopy (AFM) is a valuable tool for creating high-resolution images of cellular surfaces in air and also has the ability to image living cells in solution.11−15 It has the advantage that it shows a lateral surface view of the cell envelope, complementary to the cross-sectional view in TEM, and can show the effects of biocidal compounds as damage occurs. Using AFM and TEM, we generated high-resolution images of cell surfaces in air and in solution after (or during) exposure to PPE-Th and EO-OPE-1(C3). One goal was to confirm the mechanism of dark killing by cellular disruption proposed by Wang et al.1 by observing changes in the cell surfaces. The presence of surface aggregates on the bacterial surface after E. coli was exposed to PPE-Th supports a mechanism where the biocide causes large enough surface disruptions to kill the cells. The absence of similar aggregates for the EO-OPE-1(C3) case is also consistent with the hypothesized mechanism. In this case, the oligomger penetrates the outer cell membrane, causing channel formation that ultimately results in cell death. A series of real-time AFM images of live E. coli cells exposed to EO-OPE-1(C3) in solution shows increasing cell surface disruption with length of time after the introduction of EO-OPE-1(C3).
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METHODS
Bacterial Cultures. The antimicrobial materials used in this study were synthesized as previously reported.6,16 The exact molecular weight of the CPE polymer is not known, but the average molar mass value is estimated to be within the range of 20−30 kDa. Escherichia coli B (ATCC 11303) was obtained from the American Type Culture Collection ATCC (Manassas, VA) and grown in standard Luria broth. A fresh bacterial culture was inoculated from an overnight culture followed by ∼3 h of incubation at 37 °C to the exponential growth phase, after which the cells were collected by centrifugation. The collected cell pellet was washed twice with 10 mM phosphate-buffered saline (PBS) (138 mM NaCl and 2.7 mM KCl at pH 7.4) and then was resuspended in distilled water for AFM imaging. Electron Microscopy. Fresh bacterial cells in the exponential growth phase ((1−4) × 108 colony forming units (CFU)/mL) were incubated in a 0.85% NaCl sterile solution and various amounts of CPEs or OPEs and kept in the dark at 37 °C for 1 h before being imaged by TEM as previously described.1,3 Briefly, the cell pellets were fixed with 2% glutaraldehyde for 1 day and then stained with 1% osmium tetroxide (a lipid stain) for 1 h at room temperature. The samples were then dehydrated by sequential treatment with increasing concentrations of ethanol, embedded in resin (Spurr’s resin kit, Electron Microscopy Sciences, Hatfield, PA), sectioned, and imaged by TEM (Hitachi H7500, Tokyo, Japan). Cleaning. Glass coverslips (VWR microglass 12 mm no. 2) were placed in piranha solution (3:1 H2SO4/H2O2 stock concentrations) for 2 h. The coverslips were then rinsed with nanopure water and dried under a flow of N2. Air Imaging. Air-dried samples were prepared by pipetting 25 μL of 108 cells/mL E. coli in distilled water solution onto a cleaned glass coverslip. The cells were allowed to physisorb in a closed Petrie dish at 100% humidity (to prevent evaporation) for 1 h before being gently rinsed with 0.5 mL of nanopure water. The sample was then thoroughly dried under N2 gas. Imaging was performed using a Nanoscope IIIa atomic force microscope (AFM) in tapping mode under a constant flow of dry N2 gas using a rectangular silicon cantilever with a spring constant of 40 N/m (Veeco model RTESPAW). Standard Veeco imaging software (Nanoscope V531r117) was used to capture and analyze the images. Underwater Imaging. For underwater imaging, cleaned coverslips were first coated with Cell-Tak adhesive protein.18 The Cell-Tak solution18 was 57:2:1 NaHCO3(0.1 M)/Cell-Tak(stock)/NaOH(1
Figure 2. AFM scans of air-dried E. coli cells. (a) 5 μm × 5 μm continuous layer of E. coli cells. The scale bar is 1.0 μm. Some cells have been distorted or destroyed from the drying process (arrows). (b) 0.5 μm × 0.5 μm scan of a single E. coli cell surface. The scale bar is 100 nm. The white box marks the area of increased resolution shown in panel c. (c) 200 nm × 200 nm high-resolution scan of the cell surface. The scale bar is 50 nm. The bump features dominating the surface are interpreted as porins. (Inset) Software zoom of a single porin with a scale bar of 2.5 nm. (d) Histogram of the porin areas shown in panel c, with a peak area of 10 nm2. (Inset) Inverted binary image where the white areas were used to determine the porin areas. 10692
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created by setting the highlighted features to 1 and the nonhighlighted surroundings to 0. The binary image was analyzed in ImageJ, which output the pixel-by-pixel areas of all nonzero features. The pixel areas were then converted to physical areas for the histogram in Figure 2d.
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RESULTS/DISCUSSION Air Images: Cells Only. Figure 2a shows a 5 μm × 5 μm AFM scan of a continuous layer of air-dried E. coli cells. The
Figure 4. E. coli cells exposed to EO-OPE-1(C3). (a) 10 μm × 10 μm continuous layer of E. coli cells that has been dried after dark exposure to 30 μg/mL EO-OPE-1(C3) for 1 h. The scale bar is 1.0 μm. Intact E. coli are still visible (blue, solid arrow), but clumps of debris from cell destruction are clearly present (red, dashed arrow). The white box marks the area of a real-time zoom for panel b. (b) 500 nm × 500 nm scan of the cells in a seemingly debris-free area. The scale bar is 100 nm. Wrinkles from the drying process are also visible (arrow). (c) 200 nm × 200 nm software zoom of the exposed E. coli surface. The scale bar is 50 nm. The circular porin structures are now absent, replaced by linear, wormlike features (arrow). (d) TEM image of E. coli cells after 1 h of dark exposure to 10 μg/mL EO-OPE-1(C3). The scale bar is 500 nm. Amorphous materials outside the cells are observed (blue, solid arrow), but the cellar envelope does not show the clear disruption of the previous polymer case (red, dashed arrow).
Figure 3. E. coli cells exposed to PPE-Th. (a) 10 μm × 10 μm continuous layer of E. coli cells that has been dried after dark exposure to 30 μg/mL PPE-Th for 1 h. The scale bar is 1.0 μm. Debris from cell destruction is spread across the image (red, dashed arrow), but intact E. coli cells are still present (blue, solid arrow). (b) 2 μm × 2 μm realtime zoom of the cells in a seemingly debris-free area. The scale bar is 100 nm. (c) 500 nm × 500 nm real-time zoom of the exposed E. coli surface. The scale bar is 100 nm. (d) TEM image of E. coli cells after 1 h of dark exposure to 10 μg/mL PPE-Th. The scale bar is 500 nm. Similar to the AFM images, high-density aggregates are visible on the outer cell membrane (blue, solid arrow and red, dashed arrow). Significant disruption of the cell envelope is also visible (green, short dotted arrow, and brown, circle-line arrow).
Figure 2b is a 0.5 μm × 0.5 μm AFM scan of a bacterial surface from a sample similar to the one shown in Figure 2a. The image shows a surface containing large deformations covered by small bump features. Due to their size and shape variation, the large deformations are believed to be creases caused by the drying process. The sizes and shapes of the smaller features are consistent with porin protein channels on the E. coli outer membrane surface. These features are better seen in Figure 2c, which is a 200 nm × 200 nm scan of a part of the same region shown in Figure 2b. The bump features dominating Figure 2c are fairly regularly spaced across the image and in many cases appear to contain holes in their centers, consistent with porin molecules.24−27 This interpretation is further supported by the histogram of the nominal porin areas shown in Figure 2d, which has a peak at about 10 nm2. In very high resolution AFM images of 2D crystals of the outer membrane porin OmpG,25 the average diameter at the highest ridge around the porin channel is about 3.2 nm, giving an area of 8.0 nm2, consistent with the approximate size found here. Air Images: Cells and Biocides. Figure 3a shows a 10 μm × 10 μm image of E. coli cells which were dried after exposure to 30 μg/mL PPE-Th for 1 h under dark conditions. These images therefore show the effects of PPE-Th without lightinduced biocidal activity. In contrast to Figure 2a, many cells now appear to be physically damaged, and some parts of the image show fields of debris. Figure 3b is a 2 μm × 2 μm image
drying process concentrates the cells, producing a solid layer that is self-supporting and hence able to withstand AFM imaging forces despite weak individual attachments to the substrate. The images resemble previously reported AFM images of dried E. coli.19−23 For example, Razatos et al.23 show a similar lawn of E. coli cells. In both Razatos et al. and in Figure 2a, a majority of cells appear to have intact outer membranes, but Razatos et al. fixed their cells with glutaraldehyde, preserving the original cell shape. As we did not use any fixing agents, our cells have a flattened appearance and contain surface wrinkles and other large-scale deformations. In addition, some cells may also have been destroyed in the drying process (as evidenced by the small, thin cellular features in Figure 2a). However, the absence of fixing agents was thought to better preserve the small-scale native surface structure of the cell, an important feature when exploring the effects of the biocides. The dried cells have an average thickness of 0.8 μm and ranged in length between 2 and 5 μm. The cells have a homogeneous appearance and are relatively free of debris. 10693
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Figure 5. Underwater AFM scans of E. coli cells. (a) 3.2 μm × 3.2 μm AFM scan of E. coli cells in nanopure water. (b) A software zoom from the initial image in panel d. (c) A software zoom from the final image, panel m, 104.3 min after EO-OPE-1(C3) exposure. Scale bars for panels a−c are 500 nm. (d) Initial AFM scan before the addition of EO-OPE-1(C3). The scale bar is 1.0 μm. (e−h) Consecutive AFM scans after the addition of 30 μg/mL EO-OPE-1(C3) to the water solution surrounding the cells (blue, solid arrow). (i) The surrounding solution was refreshed with more 30 μg/ mL EO-OPE-1(C3). The image disruption in the lower left corner is a result of the biocide refreshment (red, dashed arrow). This also causes a slight downward shift in the AFM scan area. The upper area of these images corresponds to the lower region of the initial scan as the AFM drifted during the experiment. (j−m) Consecutive AFM scans after the oligomer refreshment. Images d−m have been passed through a Gaussian filter to minimize height differences. The unprocessed image is visible in the lower left inset for images d−m. Time stamps mark the time of capture in minutes from the initial scan.
same conditions as the above AFM images. In agreement with the conclusions of the above paragraph, the cells show obvious regions where the outer membrane is disrupted (arrows) but also regimes where the cell surface seems to be coated. Even though some cells show reduced cytoplasm in their interior (lighter cells), large debris fields are not present. Figure 4a shows a 10 μm × 10 μm continuous layer of E. coli cells which were dried after exposure to 30 μg/mL EO-OPE1(C3) for 1 h under dark conditions (no light-activated biocidal activity). As with PPE-Th, areas of debris are present (arrows), but they appear to be tighter aggregates, mostly clustered around cell borders, and cell surfaces are smooth, similar to the unexposed E. coli in Figure 2. Figure 4b is a 500 nm × 500 nm image of one exposed E. coli cell surface. On this scale the surface is generally smooth except for wrinkles due to drying and is similar in appearance to the surfaces of unexposed cells (Figure 2). The surface also shows small-scale features (Figure 4c), similar in size to the porins in Figure 2c. But in Figure 4c these features appear to be linear or wormlike rather than the compact circles seen in Figure 2c. They cannot therefore be identified as intact porin channels and may represent disrupted porins or possibly aggregates of OPE molecules, lipids, and porin proteins. Figure 4d is a TEM image of E. coli cells after 1 h of dark exposure to 10 μg/mL EO-OPE-1(C3). Many of the EO-OPE-1(C3) exposed cells have lost their interior cytoplasm, giving them a lighter appearance. The lack of dark surface aggregates is consistent with the AFM images. The mechanism by which EO-OPE-1(C3) kills cells thus seems very
of the cells in a seemingly debris-free area that reveals a radically different surface from the untreated cell images above. On the smallest scale the surfaces now show a roughened appearance, and on larger scales, they are marked by high “aggregate” features. These features are also seen in Figure 3c, which is a 500 nm × 500 nm scan of the same region of the exposed E. coli surface. Small aggregates dominate the surface. The porins and other surface features from Figure 2c are no longer visible. These features can be interpreted either as a disrupted outer membrane or as a coating by the PPE-Th polymer itself (or both). It is possible to estimate whether there is enough PPETh in solution to coat the surfaces of the cells. We approximate a typical cell shape as a cylinder of 1.5 μm length and 0.5 μm diameter, capped by two hemispheres of the same diameter (giving a cell with a total length 2 μm). The average area per cell is then about 2.7 μm2. For concentrations of 108 cells/cm3 and 30 μg/cm3 PPE-Th, each cell could be coated with up to 3 × 10−7 μg of PPE-Th. The density of this coating is not known, but most organic polymer solids have densities of less than 1.2 g/mL.28 This means that there is enough PPE-Th in solution to coat each cell to a depth of (3 × 10−13 g/1.2 g/cm3)/2.7 × 10−8 cm3 = 90 nm. Therefore, depending on what fraction of PPETh is absorbed, the features seen in Figure 3c could be pure polymer or disrupted outer cell membrane or some combination of these. Figure 3d is a TEM image of E. coli cells that have been exposed in the dark to 10 μg/mL PPE-Th for 1 h under the 10694
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provided a strongly attached, tightly packed cell layer without a large excess of loose cells. This method enables AFM images of cells without using fixing agents or mechanical trapping. Figure 5 shows E. coli cells in nanopure water as 30 μg/mL EO-OPE-1(C3) is introduced into the surrounding solution. (Attempts to introduce PPE-Th solutions caused tip fouling and were not successful.) Figure 5a is a 2 μm × 2 μm AFM control image of E. coli cells in nanopure water alone, without biocide. The image resembles earlier published images of E. coli in solution,31,32 with visible surface features and no artifacts or debris. Figure 5b is a 500 nm × 500 nm image of an E. coli cell before the biocide had been added at the beginning of the experiment, and Figure 5c is a 500 nm × 500 nm image of nearby E. coli cells 104.3 min after the biocide had been added, at the end of the experiment. Together, Figure 5b,c shows the increased cell surface roughness that results from exposure to 30 μg/mL EO-OPE-1(C3) in solution. Figure 6d−m shows the AFM time series over the course of that exposure. (These images have been passed through a Gaussian filter to minimize height differences.) As time progresses, cell surfaces demonstrate visible changes and increased roughness. At the 46.4 min mark, the solution within the fluid cell was refreshed with more 30 μg/mL EO-OPE-1(C3). After the solution refresh, E. coli show a dramatic increase in cell surface roughness, but no internal cell contents are exposed during this series.
Figure 6. Hypothesized mechanism for the dark antimicrobial action of PPE-Th and EO-OPE-1(C3). (a) Structure of unexposed cell envelope with lipid bilayers (orange lipid heads with gray tails), lipopolysaccharides (gray), peptidoglycan (dark red), membrane proteins (blue, dark green, and dark red), and porins (light green). (b) The PPE-Th polymer (yellow) forms micellelike aggregates with the outer membrane of the E. coli cells. The formation of these polymer aggregates disrupts the integrity of the membrane, leading to cell death. (c) The oligomer (yellow) is a much smaller molecule and can associate with, or perhaps penetrate, the outer cell membrane of E. coli. Instead of forming visible aggregates, the oligomer forms pores that disrupt the chemical gradient, leading to cell death.
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CONCLUSIONS The changes to the cell morphology caused by the PPE-Th polymer resemble (at least superficially) the effects of antimicrobial peptides such as NK-2.11,21,33 In both cases, aggregates of polymer and cellular material bind to the E. coli cell wall envelope, making the entire surface appear rougher in the AFM height images. However, the cell-killing mechanism of the polymer is not a simple removal of the outer membrane. Images obtained by Amro et al.19 show the effect on E. coli caused by EDTA, and the pattern of damage (with holes appearing on an otherwise smooth surface) clearly differs from the observed effects of the PPE-Th polymer on the cells. A proposed dark killing mechanism is illustrated in Figure 6a. The polymer forms aggregates with the bacterium’s outer membrane, causing damage via partial emulsification with large surface disruptions. When enough disruption of the cell surface has occurred, the cellular contents are released.4 The EO-OPE-1(C3) oligomer does not coat the cell surface, but changes are still apparent in the organization of proteins and, presumably, other outer-membrane components. This observation is consistent with Figure 6b, in which the EO-OPE1(C3) oligomer is a much smaller biocide that can associate with the outer membrane, causing the reorganization of membrane components without wholesale loss of integrity.5,7 Using the AFM, we observed changes in E. coli surfaces caused by exposure to the biocides PPE-Th polymer and EOOPE-1(C3) oligomer. Control images established that the dried E. coli cells can be imaged with enough resolution to identify porins on the surface. The PPE-Th polymer causes the formation of large aggregates on the surfaces of dried cells. The high aggregate density relative to the polymer concentration in solution favors a primarily PPE-Th composition. The aggregates obscure surface features such as the porins, indicating possible major disruption or even partial removal of the outer membrane. This disruption is the assumed dark killing mechanism of the PPE-Th polymer. After EO-OPE1(C3) exposure, cellular debris is apparent but the surface
different than the PPE-Th polymer. This is consistent with earlier findings.1 Imaging Live Cells by AFM. Imaging live bacterial cells by AFM has often proven to be challenging. The cells usually do not adhere to glass or mica substrates strongly enough to keep them stationary under tip forces. Several different techniques have been developed to address this issue.18 One method, developed in 1995 by Kasas and Ikai,29 utilizes isopore polycarbonate membranes with pore sizes similar to the cell size needed to trap bacteria mechanically. Once trapped, the top portion of a cell can be imaged by AFM without risk of bacterial movement. A few groups have adopted this technique,13,30 or employed close variants,18 for imaging spherical cells. Another approach uses treated surfaces to immobilize cells, such as glass treated with poly(ethyleneimide),15 glass treated with poly-L-lysine,14 or mica treated with poly-L-lysine.31,32 Some authors23 used glutaraldehyde fixation to preserve cell shape. We were able to image fields of live cells by (a) coating a cover glass with adhesion protein Cell-Tak18 to increase cell− surface attachment and (b) allowing approximately two-thirds of the initial droplet to evaporate, thus concentrating the cells into a self-supporting packed layer. Because the evaporation process would concentrate any dissolved salts present, the cells were deposited from distilled water. Attempts to pack the surface by simply using a high cell concentration were not successful, either because the cells did not attach strongly enough or because a large number of loosely bound cells fouled the cantilever. Allowing the solution to partially evaporate 10695
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Fernandes, M. X.; Castanho, M. A. R. B. Escherichia coli cell surface perturbation and disruption induced by antimicrobial peptides bp100 and pepr. J. Biol. Chem. 2010, 285, 27536−27544. (12) Dufrêne, Y. F. Application of atomic force microscopy to microbial surfaces: From reconstituted cell surface layers to living cells. Micron 2001, 32, 153−165. (13) Dufrêne, Y. F.; Boonaert, C. J. P.; van der Mei, H. C.; Busscher, H. J.; Rouxhet, P. G. Probing molecular interactions and mechanical properties of microbial cell surfaces by atomic force microscopy. Ultramicroscopy 2001, 86, 113−120. (14) Vadillo-Rodriguez, V.; Beveridge, T. J.; Dutcher, J. R. Surface viscoelasticity of individual gram-negative bacterial cells measured using atomic force microscopy. J. Bacteriol. 2008, 190, 4225−4232. (15) Velegol, S. B.; Logan, B. E. Contributions of bacterial surface polymers, electrostatics, and cell elasticity to the shape of afm force curves. Langmuir 2002, 18, 5256−5262. (16) Zhou, Z.; Corbitt, T. S.; Parthasarathy, A.; Tang, Y.; Ista, L. K.; Schanze, K. S.; Whitten, D. G. end-only” functionalized oligo(phenylene ethynylene)s:Synthesis, photophysical and biocidal activity. J. Phys. Chem. Lett. 2010, 1, 3207−3212. (17) Nanoscope software 531r1; Veeco Instruments Inc, 2004. (18) Louise Meyer, R.; Zhou, X.; Tang, L.; Arpanaei, A.; Kingshott, P.; Besenbacher, F. Immobilisation of living bacteria for afm imaging under physiological conditions. Ultramicroscopy 2010, 110, 1349− 1357. (19) Amro, N. A.; Kotra, L. P.; Wadu-Mesthrige, K.; Bulychev, A.; Mobashery, S.; Liu, G.-y. High-resolution atomic force microscopy studies of the escherichia coli outer membrane: Structural basis for permeability. Langmuir 2000, 16, 2789−2796. (20) Eaton, P.; Fernandes, J. o. C.; Pereira, E. l.; Pintado, M. E.; Xavier Malcata, F. Atomic force microscopy study of the antibacterial effects of chitosans on escherichia coli and staphylococcus aureus. Ultramicroscopy 2008, 108, 1128−1134. (21) Meincken, M.; Holroyd, D. L.; Rautenbach, M. Atomic force microscopy study of the effect of antimicrobial peptides on the cell envelope of escherichia coli. Antimicrob. Agents Chemother. 2005, 49, 4085−4092. (22) Peng, L.; Yi, L.; Zhexue, L.; Juncheng, Z.; Jiaxin, D.; Daiwen, P.; Ping, S.; Songsheng, Q. Study on biological effect of la3+ on escherichia coli by atomic force microscopy. J. Inorg. Biochem. 2004, 98, 68−72. (23) Razatos, A.; Ong, Y.-L.; Sharma, M. M.; Georgiou, G. Molecular determinants of bacterial adhesion monitored by atomic force microscopy. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 11059−11064. (24) Schabert, F. A.; Engel, A. Reproducible acquisition of escherichia coli porin surface topographs by atomic force microscopy. Biophys. J. 1994, 67, 2394−2403. (25) Mari, S. A.; Köster, S.; Bippes, C. A.; Yildiz, Ö .; Kϋhlbrandt, W.; Muller, D. J. Ph-induced conformational change of the i2̂ -barrelforming protein ompg reconstituted into native e. Coli lipids. J. Mol. Biol. 2010, 396, 610−616. (26) Meroueh, S. O.; Bencze, K. Z.; Hesek, D.; Lee, M.; Fisher, J. F.; Stemmler, T. L.; Mobashery, S. Three-dimensional structure of the bacterial cell wall peptidoglycan. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 4404−4409. (27) Schabert, F. A.; Henn, C.; Engel, A. Native escherichia coli ompf porin surfaces probed by atomic force microscopy. Science 1995, 268, 92−94. (28) Scientific Instrument Services, I. Santovac 5 polyphenyl ether vacuum pump fluid. http://www.sisweb.com/vacuum/sis/satovc5p. htm, Feb. 12, 2014. (29) Kasas, S.; Ikai, A. A method for anchoring round shaped cells for atomic force microscope imaging. Biophys. J. 1995, 68, 1678−1680. (30) Touhami, A.; Jericho, M. H.; Beveridge, T. J. Atomic force microscopy of cell growth and division in staphylococcus aureus. J. Bacteriol. 2004, 186, 3286−3295. (31) Fantner, G. E.; Barbero, R. J.; Gray, D. S.; Belcher, A. M. Kinetics of antimicrobial peptide activity measured on individual
remains largely intact. Porins are no longer identifiable as the surface is dominated by linear bump features, likely aggregated protein or protein-lipid-OPE complexes. This observation suggests that the mechanism for oligomer dark killing involves surface association or possible insertion of EO-OPE-1(C3) into the cell envelope. In solution, the oligomer caused the surface of E. coli to increase in roughness over time. Overall, our observations support the dark killing mechanisms put forward by Wang et al.1,4
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The research reported here was supported by the Defense Threat Reduction Agency-Joint Science and Technology Office for Chemical and Biological Defense (grant no. HDTRA1-11-10004 to D.J.K. and D.G.W.). Special thanks go to Kirk Schanze, Department of Chemistry, University of Florida (Gainsville, FL) and Zhijun Zhou for their generous gifts of PPE-Th and EO-OPE-1(C3), respectively.
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